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Research Open Access A novel and ancient group of type I keratins with members in bichir, sturgeon and gar Michael Schaffeld*1, Mark Haberkamp1, Sonja Schätzlein2, Sebastian Neumann1 and Christian Hunzinger3

Address: 1Institute of Zoology, Johannes-von-Müller-Weg 6, Johannes Gutenberg University, D-55099 Mainz, Germany, 2Dept. of Gastroenterology, Medical School Hannover, Carl Neuberg Str. 1, K11, E01, R1400, 30629 Hannover, Germany and 3Merck KGaA, Central Services Analytics, Central Product Analytics/Bioanalytics, Frankfurter Str. 250, D-64293 Darmstadt, Germany Email: Michael Schaffeld* - [email protected]; Mark Haberkamp - [email protected]; Sonja Schätzlein - schaetzlein.sonja@mh- hannover.de; Sebastian Neumann - [email protected]; Christian Hunzinger - [email protected] * Corresponding author

Published: 6 June 2007 Received: 22 December 2006 Accepted: 6 June 2007 Frontiers in Zoology 2007, 4:16 doi:10.1186/1742-9994-4-16 This article is available from: http://www.frontiersinzoology.com/content/4/1/16 © 2007 Schaffeld et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract 1. Background: Vertebrate epithelial cells typically express a specific set of keratins. In teleosts, keratins are also present in a variety of mesenchymal cells, which usually express vimentin. Significantly, our previous studies revealed that virtually all known teleost keratins evolved independently from those present in terrestrial vertebrates. To further elucidate the evolutionary scenario that led to the large variety of keratins and their complex expression patterns in present day teleosts, we have investigated their presence in bichir, sturgeon and gar. 2. Results: We have discovered a novel group of type I keratins with members in all three of these ancient ray-finned fish, but apparently no counterparts are present in any other vertebrate class so far investigated, including the modern teleost fish. From sturgeon and gar we sequenced one and from bichir two members of this novel keratin group. By complementary keratin blot-binding assays and peptide mass fingerprinting using MALDI-TOF mass spectrometry, in sturgeon we were able to assign the sequence to a prominent protein spot, present exclusively in a two-dimensionally separated cytoskeletal preparation of skin, thus identifying it as an epidermally expressed type I keratin. In contrast to the other keratins we have so far sequenced from bichir, sturgeon and gar, these new sequences occupy a rather basal position within the phylogenetic tree of type I keratins, in a close vicinity to the keratins we previously cloned from river lamprey. 3. Conclusion: Thus, this new K14 group seem to belong to a very ancient keratin branch, whose functional role has still to be further elucidated. Furthermore, the exclusive presence of this keratin group in bichir, sturgeon and gar points to the close phylogenetic relationship of these ray- finned fish, an issue still under debate among taxonomists.

Background from typeI/II heterodimers. The keratins are members of In vertebrates the cytoskeleton of epithelial cell types is the large multigene family of intermediate filament pro- typically reinforced by a specific set of type I and type II teins (IFproteins) of which they form by far the most com- keratins that assemble into 10 nm thick filaments formed plex group. In human, 53 of the hitherto nearly 70

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identified IF protein genes code for keratins [1-4] that are Results and discussion expressed in tissue and developmental specific patterns. Novel type I keratin sequences from bichir, sturgeon and Without including human hair and nail forming keratins, gar the number of keratin genes found in teleost fish is com- Only recently we have discovered that a rather ancient and parably high, but in contrast to human and other tetrap- distant group of keratin-related sequences, the extracellu- ods, teleost fish possess a large excess of type I keratin larly secreted thread keratins TKα and TKγ, are not only genes [5-7]. By analysing the molecular evolution of kerat- present in hagfish (the assumedly most ancient vertebrate ins, as well as the evolution of their expression patterns in group), but also in lamprey, teleosts and amphibians. lower vertebrates, we want to further elucidate the sce- This provided major clues relating to keratin evolution in nario and probable evolutionary forces that led to this vertebrates, but also pointed to a more general role of this extraordinary variety of keratins in vertebrates. previously considered highly specific IF protein group in vertebrates [28]. By combination of RT-PCR experiments Our investigations of the keratin systems in lamprey, and cDNA library screening (for details see Methods), shark, trout, zebrafish, carp, goldfish and lungfish have so from sturgeon and gar we have now isolated one and from far revealed that type I and type II keratins are apparently bichir two cDNA sequences that, according to our phylo- present in all classes of vertebrates and that the various genetic analysis, code for members of another novel kera- keratins can generally be subdivided into the "E" keratins, tin group (Fig. 1, Table 1). The 1858 bp long cDNA clone expressed in epidermal keratinocytes and other stratified we isolated from the sturgeon cDNA library (abak14; epithelia, and those appearing in cells forming simple epi- [EMBL: AJ493259]) contains the complete coding thelia, thus named "S" keratins [8-19]. Nevertheless, our sequence for a type I keratin of 46759Da (431 amino data based on cDNA sequence analysis, followed by thor- acids) and a calculated pI of 5.1, which we now term ough phylogenetic analyses [13-18] as well as the studies AbaK14 (from Acipenser baeri keratin). However, the cor- based on the recently available genome data from man responding sequences we have so far obtained from bichir and teleost fish [1,2,5-7], strongly support the view of and gar are still incomplete. A 1369 bp long clone largely independent origins of the keratin genes found in (pseK14a; [EMBL: AM419452]) isolated from the bichir fish and man. According to our present data, solely the cDNA library encodes a type I keratin that we term typical "S" keratin pair K8 and K18 can at least be found PseK14a (Polypterus senegalus keratin). It still lacks a por- in all gnathostomian vertebrate groups, indicating the tion of its head encoding sequence in addition to its 5' unique and general importance of these "ancient" kerat- UTR. By RT-PCR using degenerate primers, from bichir we ins. In contrast to other vertebrates investigated so far, in additionally recovered a 908 bp long cDNA sequence modern teleost fish keratins, including K8 and K18, in (pseK14b; [EMBL: AM419453]), comprising almost the addition to their typical IF epithelial appearance show a complete rod encoding segment of a second K14 counter- widespread IF occurrence in mesenchymally derived cells part in this species (PseK14b). In addition, we found five and tissues, such as fibroblasts, chondrocytes and blood further incomplete cDNA clones that apparently encode vessel endothelia (for review see [11]). The latter in the different variants of PseK14a (not shown here). The latter non-teleost vertebrates usually do not express keratins but only slightly vary in DNA sequence, from 0.1 – 3.1% the type III IF protein vimentin [20-26]. To further trace (amino acid variance of 0.4 – 4.9%). In a similar way we the origin of the different "E" and "S" keratins as well as were also able to amplify a cDNA fragment encoding the the evolution of the mesenchymal keratin expression in rod domain of a K14 counterpart in gar, which we term teleosts, we have investigated the keratin systems in a LocK14 (from Lepisosteus oculatus keratin). By RACE-PCR bichir, a sturgeon and a gar that are believed to represent we additionally recovered its tail encoding sequence and the most ancient groups of the extant ray-finned fish. In 3' UTR. Its assembled sequence (lock14; [EMBL: the course of these studies, from all three species we AM419454]) overall comprises 1207 bp, but still lacks the obtained sequences that apparently belong to a novel complete head encoding segment in addition to the sec- branch of type I keratins, without counterparts in any tion coding for the first seven residues of the rod domain. other vertebrate group investigated so far, including the teleost fish. Here we present and discuss their sequences Subsequent mining of the available genome and EST data- as well as their phylogenetically relationships to the other bases for K14 counterparts in other vertebrates such as tel- members of the type I keratin subfamily, which may also eosts, amphibians, birds and mammals, so far has not yet provide clues to the early evolution of ray-finned fish. The yielded any matches, suggesting that this keratin group latter is still strongly debated among taxonomists, may only be present in the ancient groups of ray- finned whether on the basis of molecular or morphological data fish. We only found two matches encoding K14 of (for an overview see [27]). another sturgeon, notably Acipenser transmontanus (white sturgeon; [EMBL: DR975435, DR975694]), which both stem from a skin-derived cDNA library.

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SequenceFigure 1 comparison of K14 from bichir, sturgeon and gar Sequence comparison of K14 from bichir, sturgeon and gar. Multiple alignment of the keratin 14 (K14) sequences we obtained from bichir, sturgeon and gar. Thick black lines mark the four helical subdomains (coils 1A to 2B), which are typical for the central rod domain of all known IF-proteins. Asterisks indicate identical amino acids; double dots indicate a high and single dots a lower degree of amino acid conservation. Pse, Polypterus senegalus (bichir); Aba, Acipenser baeri (sturgeon); Loc, Lepisosteus oculatus (gar). Note that only AbaK14 comprises the complete amino acid sequence. From PseK14a at least a sec- tion and from LocK14 the complete head sequence is still missing. From PseK14b we still have to recover both, the complete head and tail sequence.

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Table 1: Properties of the isolated cDNA clones encoding K14 from bichir, sturgeon and gar.

cDNA clone Keratin EMBL accession number Size of cDNA (bp) Number of encoded amino acids Mr (Da) pI

abaK14 AbaK14 AJ493259 1858 431 467594) 5.14) pseK14a PseK14a AM419452 1369 3631) -- pseK14b PseK14b AM419453 908 3022) -- locK14 LocK14 AM419454 1207 3513) --

Pse, Polypterus senegalus (bichir); Aba, Acipenser baeri (sturgeon); Loc, Lepisosteus oculatus (gar); 1) a portion of the head domain is still missing; 2) head and tail domain are still missing; 3) complete head domain is still missing; 4) as predicted from the cDNA sequence.

Biochemical identification of K14 in sturgeon assign a single protein spot to the amino acid sequence To analyse the general set of keratins expressed in stur- derived from the cloned abak14 cDNA sequence. The geon, we extracted the cytoskeletal proteins from different matching spot was solely found as a major component in tissues, including skin, liver, intestine, stomach and gill, the cytoskeletal preparation of skin (Fig. 2a), in which it separated them by 2D-PAGE and subsequently analysed was firmly identified as a type I keratin in the CKBB assay the patterns by CKBB assays and immunoblotting (results (Fig. 2a"). It showed a positive reaction with the anti- for skin, stomach and intestine are shown in Fig. 2). The trout-keratin antiserum GPpoly (data not shown), which major spots were additionally analysed by peptide mass we previously introduced as a general keratin marker in fingerprinting (PMF) to reveal similarities between identi- fish [8,10-15,19]. The two-dimensional position of this fied keratin spots and to assign them to the sequences we protein spot fits the theoretical values calculated from the obtained from the sturgeon by cDNA library screening. In sequence of AbaK14 (see above and Table 1). Peptide the course of these investigations we were clearly able to mass fingerprint (PMF) analysis of this protein yielded 29 matching peptide masses for AbaK14, of which 26 were specific for AbaK14 in comparison to the other six type I keratins we so far sequenced from sturgeon. Overall amino acid sequence coverage for AbaK14 was 61%.

Molecular evolution of keratins – the ancient origin of K14 To infer the phylogenetic relationships of the novel K14 group to the other currently known type I keratins, by different methods we thoroughly analysed a comprehensive data set of 118 polypeptides, including the type I keratins from lancelet, lamprey, shark, bichir, sturgeon, gar, zebrafish, trout, Xenopus, lungfish and man (for further details see Methods; the available accession numbers of the employed sequences are listed in Fig. 3). Most of the fish sequences stem from our own data, notably those BiochemicalFigure 2 identification of K14 in sturgeon from lamprey, shark, bichir, sturgeon, gar, trout and lung- Biochemical identification of K14 in sturgeon. 2D- fish in addition to K8 and K18 from zebrafish [13- PAGE of cytoskeletal proteins extracted from sturgeon skin 18,23,29,30]. We rooted the trees with the type I keratin (a), stomach (b) and intestine (c). Isoelectric focusing (IEF) sequences available from the lancelets, which are believed was used in the first dimension, in the second dimension we to represent the most ancient group of living chordates applied SDS-PAGE. Bovine serum albumin (B) and rabbit α- [31]. In general we received the same basal tree topology actin (A) were added to the samples as marker proteins. E, when applying Neighbor Joining (NJ), Maximum Likeli- epidermal keratins; S, simple keratins. (a-c) Coomassie Blue hood (ML) or Bayesian (B) methods for our phylogenetic stained gels, showing the polypeptide patterns, which were analysis. The phylogenetic tree shown in Fig. 4 is based on subsequently used for complementary keratin blot-binding (CKBB) assays. (a'-c') CKBB test employing biotinylated Bayesian inference and clearly shows that the K14 human keratin K18, thereby identifying type II keratins (II). sequences form a separate and basal branch within the (a"-c") CKBB assay using biotinylated human keratin K8 type I keratins, phylogenetically close to the sequences we which identifies type I keratins (I). Note that the K14 spot is obtained from the river lamprey. They even branch off not present as a major component in intestine or stomach prior to the twig formed by the gnathostomian K18 and that both tissues express a mixture of E and S keratins. sequences, that apparently emerged before the separation of cartilaginous and bony fish [13-15]. This ancestral ori-

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gin of the K14 twig together with the assumedly exclusive a sister group to the teleost fish and that bichirs represent presence of K14 in ancient ray-finned fish (see above) the most ancient group of ray-finned fish (for review see raises the question as to the complementary binding part- [27]). Since our current studies do not contribute to the ner(s) of this keratin group. So far, analysis of bichir and resolution of this problem, the keratin system in bowfin sturgeon has not revealed a group of type II keratins occu- should be investigated, which might shed more light on pying a similar basal position equivalent to the K14 this issue. sequences in the type I keratin tree [8,29,30]. Moreover, the suggested restriction of K14 sequences to bichir, stur- Furthermore, the phylogenetic tree illustrated in Fig. 4 geon and gar indicates the close phylogenetic relationship suggests that most of the type I keratins from ray-finned of these fish groups, an issue still under debate among tax- fish evolved independently from those present in lung- onomists. Based on recent molecular data it can be con- fish, frog or man and that very early in actinopterygian cluded that sturgeons, gars and bowfin together may form evolution a gene duplication gave rise to at least two dif-

AccessionFigure 3 numbers Accession numbers. EMBL accession numbers of the type I keratin sequences we used for phylogenetic inference. *For three Xenopus tropicalis type I keratin sequences the Ensembl database gene IDs are given.

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MolecularFigure 4 evolution of type I keratins Molecular evolution of type I keratins. Phylogenetic tree based on Bayesian inference, illustrating the relationships of the K14 sequences from bichir, sturgeon and gar to the other type I keratins known from vertebrates. The tree was rooted with the lancelet type I keratin sequences. It clearly shows that the K14 sequences form a separate branch (boxed in violet) close to the sequences we cloned from the river lamprey. They even branch off prior to the twig formed by the gnathostomian K18 sequences (boxed in green) that apparently emerged before the separation of cartilaginous and bony fish. The tree, furthermore, suggests that most of the ray-finned fish type I keratins (boxed in blue) evolved independently from those present in lungfish, frog or man and that early in actinopterygian evolution gene duplications already gave rise to at least two different type I keratin groups with members in both, ancient and modern ray-finned fish. Importantly, within the tetrapod lineage the Bayesian analysis revealed four highly supported keratin subgroups, each with members in both, frog and man (encircled by dotted lines and coloured orange). Bar, 0.1 substitutions per site.

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ferent type I keratin branches, each with members in both keratins present in skin may play an important role in the ancient and modern ray- finned fish (see the boxed ray- transition of vertebrates from water- to land- living ani- fins twig in Fig. 4). Compared to our recent analyses [14- mals. Here we present a novel group of epidermal type I 18,28], we have now included additional type I keratin keratins, which we termed keratin 14 and so far have only sequences from zebrafish and Xenopus; therefore, in this found them in the basal groups of living ray-finned fish, study the K18 branch shows a more complex branching notably bichir, sturgeon and gar. Our phylogenetic analy- pattern, not allowing a clear identification of the authen- sis revealed a rather basal position of this keratin group in tic K18 counterparts in the different vertebrates solely on the tree of type I keratin evolution. The keratin 14 group the basis of their position in the tree. However, our previ- even emerged prior to the gnathostomian K18 sequences, ous identification of K18 in shark, bichir, sturgeon, trout which together with its binding partner K8 and with the and zebrafish was additionally based on its typical occur- exception of the recently discovered thread keratins in tel- rence in simple epithelia, such as liver hepatocytes or eosts and amphibians, was hitherto considered as most intestinal mucosal epithelium, corresponding to the situ- ancient group of gnathostomian keratins. Future analyses ation in man and other tetrapods [10,12-16,19,29]. Nev- will hopefully shed more light on the expression and ertheless, these "functional K18 counterparts" identified functional role of K14 in the fish epidermis and clarify the in the different vertebrate groups have to be considered as identity of its type II keratin binding partner. paralogous protein sequences. The branching pattern of the K18 twig supports the suggestion that early as well as Methods more recent duplication events led to the various K18 Preparation of tissues and cytoskeletal proteins related genes. Future analysis of their expression patterns Sturgeons (Acipenser baeri) were purchased from a local may provide further clues for possible functions of these hatching farm (Fischzucht Rhönforelle, Gersfeld, Ger- K18 relatives. many), and gars (Lepisosteus oculatus) from a local pet shop (Fauna Exotica, Mainz-Kostheim, Germany). The Compared to the evolution of type II keratins, the phylo- bichirs (Polypterus senegalus) were a gift from Dr. Latz genetic tree of type I keratins appears rather complex and (Institute of Zoology, University of Mainz). Within the several nodes cannot be resolved. Moreover, in teleosts scope of this study we employed one specimen for each the number of detected type I keratin genes virtually tri- species. Animals were killed by cutting the neck or the tail ples the number of those coding for type II keratins [5-7], artery after MS 222 narcosis (0.5 g/l). For further proce- in contrast to the almost equal number of type I and type dures, the tissues were excised and used immediately or II keratin genes detected in the tetrapod genomes. How- snap frozen according to [8]. Cytoskeletal proteins were ever, when including the hair- and nail-forming keratins extracted as described in [8]. the total number of keratin genes in tetrapods is clearly higher than in teleost fish [1-4]. A group of two sequences, Electrophoresis, immunoblotting, CKBB, PMF and one from zebrafish, the other from Xenopus, also occupy a immunofluorescence microscopy rather basal position in the type I keratin tree, directly Two-dimensional polyacrylamide gel electrophoresis branching off between the K14 and K18 twig. But it has to (2D-PAGE), complementary keratin blot-binding (CKBB) be taken into consideration that both sequences stem assays, Western blotting and indirect immunofluores- from genomic DNA sequencing and may only represent cence microscopy were essentially performed as described non-active "relics" of ancient keratin genes. From the cur- in [8]. For immunoblotting we used 10% milk powder as rent Bayesian phylogenetic analysis (but not from our NJ the blocking reagent and the antibody incubations were and ML analysis) another phenomenon emerged for the performed overnight at 8°C. Peptide mass fingerprinting first time: There are now four highly supported keratin (PMF) using matrix-assisted laserdesorption/ionization subgroups in the tetrapod twig, each with members in time-of-flight mass spectrometry (MALDI-TOF MS) was both, frog and man (in Fig. 4 encircled by dotted lines). performed as described in [16,18]. The mass tolerance for Moreover, the tree topology indicates that the human hair matching fragments was set to ≤ 100 ppm. keratins may have emerged prior to these radiation events. Preparation of RNA Conclusion Total RNA from bichir was extracted according to a proto- The type I and type II keratins represent the two most col modified from [32], including a GTC extraction and complex and abundant groups of intermediate filament subsequent sedimentation of RNA by ultracentrifugation (IF) proteins among vertebrates and their structure, func- through a dense cushion of caesium chloride (detailed tion and extraordinary variety cannot be completely protocol given in [8]). For purification of sturgeon RNA understood without phylogenetic considerations. There- we applied a protocol that included consecutive steps of fore, we investigated the keratin systems in more ancient guanidinium thiocyanate (GTC) homogenizations, acidic representatives of the vertebrate lineage. In particular, the phenol/chloroform extractions and precipitations. The

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final RNA precipitation was accomplished with 8 M LiCl. Jones-Taylor-Thornton substitution model of amino acid Gar RNA was prepared using the GeneMATRIX Universal evolution (JTT [35]). Furthermore, the model applied for RNA Purification Kit purchased from Roboklon (Ger- the ML and Bayesian analyses included observed amino many) according to the suppliers' instructions. Isolation acid frequencies (F), estimated proportion of invariant of mRNA was generally performed with the "PolyATract®- sites (I), and estimation of among-site rate variation for mRNA isolation system" from Promega. the remaining sites according to a gamma distribution (G) that was set to four rate categories. For the NJ analysis we RT-PCR analyses used the programs PROTDIST and NEIGHBOR of the Applying 0.5 to 1.0 µg of total RNA, RT-PCR was per- Phylogeny Inference Package (PHYLIP, version 3.6 b formed either using the Superscript II reverse transcriptase [36]). The reliability of the tree topology was then tested from Invitrogen (RT for 1 h at 42°C), followed by stand- by bootstrap analysis [37] with 100 replications, using the ard PCR (Taq Polymerase, Invitrogen) or the QIA- PHYLIP programs seqboot, protdist, neighbor and con- GEN®OneStep RT-PCR Kit (RT for 0.5 h at 50°C). PCR was sensus. The ML analysis was conducted with the PHYLIP- done for 30–35 cycles and primer specific annealing tem- like interface PHYML [38]. For bootstrapping we gener- peratures. We used different combinations of degenerate, ated 100 pseudo data sets. The Bayesian inference analysis IF-specific primers, as previously described [8] (notably was performed with MRBAYES 3.1 [39] with the frequen- primer numbers P4, P5, P8, P9, P10). 3' RACE-PCR was cies fixed to the Jones frequencies. In each of the two par- accomplished using an oligodT-19mer as downstream allel runs four Markov chains (one hot and three cold and sequence-specific oligos as upstream primers. For gel chains) were run simultaneously for 3,000,000 genera- extraction of PCR products we employed the QIAquick tions, starting with random trees. Sampling from the trees Gel Extraction Kit (Qiagen) or the GeneMATRIX DNA was set to every 10th generation. Under these conditions Purification Kit AGAROSE-OUT (Roboklon). Cloning of the average value for the deviation of split frequencies the isolated DNA fragments was accomplished either with reached a value < 0.01. The burn-in was set to 115000, the TOPO-TA Cloning Kit (Invitrogen), the StrataClone™ based on the stationary phase. All consensus trees were PCR Cloning Kit (Stratagene) or the pGEM-T Easy vector drawn using TREEVIEW version 1.6.6 [40]. kit (Promega). All kits were used according to the instruction manual. Primers were synthesized by Roth and Abbreviations Sigma-Ark, respectively. Nucleotide sequencing was per- CKBB Complementary keratin blot-binding formed on both strands using the Taq Dye Deoxy Termi- nator system. The subsequent gel run was done by MALDI Matrix-assisted laser desorption/ionisation commercial services (Genterprise, Germany). TOF Time-of-flight cDNA library construction and screening According to the supplier's instructions we constructed λ- MS Mass spectrometry phage cDNA libraries (ZAP-Express®, Stratagene) from sturgeon and bichir, respectively, in each case using 5 µg PAGE Polyacrylamide gel electrophoresis of mRNA purified from a mixture of tissues, including skin, eyes, brain and internal organs. Clones were isolated PMF Peptide mass fingerprint using the fish keratin-specific antiserum GPpoly [8] and digoxygenin-labeled cDNA probes derived from obtained 2D Two-dimensional RT-PCR fragments. Digoxygenin labeling was either performed using the DIG-High Prime Kit from Roche, Competing interests according to the instruction manual or by standard PCR, The author(s) declare that they have no competing inter- using digoxygenin labeled nucleotides from Roche. ests.

Sequence analyses and phylogenetic inference Authors' contributions For database searches, we employed the Basic Local Align- Mark Haberkamp performed the sequencing work in stur- ment Search Tool (BLAST [33]) of the National Center for geon and part of the biochemical work in sturgeon, Biotechnology Information (NCBI) and of the Ensembl including the analysis of the provided MALDI-TOF data. Genome Browser. The multiple sequence alignments were Sonja Schätzlein did most of the cloning experiments in performed with ClustalX version 1.8 [34] using default bichir. All experiments were accomplished while both gap penalties. When necessary, the alignment was edited authors were members of our group in Mainz. Sebastian by hand. The final alignment was analysed by Neighbor Neumann sequenced K14 from gar. Christian Hunzinger Joining (NJ), Maximum Likelihood (ML) and Bayesian performed the peptide mass fingerprint experiments and methods (B). All analyses were conducted under the assisted the analysis of the MALDI-TOF data while he was

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in the employ of ProteoSys AG, Carl-Zeiss-Str. 51, 55129 18. Schaffeld M, Bremer M, Hunzinger C, Markl J: Evolution of tissue- specific keratins as deduced from novel cDNA sequences of Mainz, Germany. Michael Schaffeld supervised the con- the lungfish Protopterus aethiopicus. Eur J Cell Biol 2005, ception of experiments, carried out part of the molecular 84:363-377. 19. García DM, Bauer H, Dietz T, Schubert T, Markl J, Schaffeld M: Iden- and biochemical studies, performed the complete phylo- tification of keratins and analysis of their expression in carp genetic analyses, including sequence alignment and tree and goldfish: comparison with the zebrafish and trout keratin catalog. Cell Tissue Res 2005, 322(2):245-56. reconstruction, and wrote the manuscript. All authors 20. Herrmann H, Fouquet B, Franke WW: Expression of intermediate read and approved the final manuscript. filament proteins during development of Xenopus laevis . I. cDNA clones encoding different forms of vimentin. Develop- ment 1989, 105:279-298. Acknowledgements 21. Lazarides E: Intermediate filaments: a chemically heterogene- We thank Prof. Dr. Harald Herrmann for providing the recombinant human ous, developmentally regulated class of proteins. Annu Rev Bio- chem 1982, 51:219-250. keratins 8 and 18, Simon Höffling for additional sequencing work, Nicole 22. Schaffeld M, Herrmann H, Schultess J, Markl J: Vimentin and desmin Tappe for additional biochemical experiments and Prof. Dr. J. Robin Harris of a cartilaginous fish, the shark Scyliorhinus stellaris : for proof reading of the manuscript. We especially thank Prof. Dr. Jürgen Sequence, expression patterns and in vitro assembly. Eur J Cell Biol 2001, 80:692-702. Markl for initiating and continuously supporting this work and critical read- 23. Schultess J: Molekulare Evolution der Intermediärfilament- ing of the manuscript. Financially aided by a grant to J.M. from the Deutsche Proteine des Flussneunauges Lampetra fluviatilis. 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